HCK induces macrophage activation to promote renal inflammation and fibrosis via suppression of autophagy

Renal inflammation and fibrosis are the common pathways leading to progressive chronic kidney disease (CKD). We previously identified hematopoietic cell kinase (HCK) as upregulated in human chronic allograft injury promoting kidney fibrosis; however, the cellular source and molecular mechanisms are unclear. Here, using immunostaining and single cell sequencing data, we show that HCK expression is highly enriched in pro-inflammatory macrophages in diseased kidneys. HCK-knockout (KO) or HCK-inhibitor decreases macrophage M1-like pro-inflammatory polarization, proliferation, and migration in RAW264.7 cells and bone marrow-derived macrophages (BMDM). We identify an interaction between HCK and ATG2A and CBL, two autophagy-related proteins, inhibiting autophagy flux in macrophages. In vivo, both global or myeloid cell specific HCK-KO attenuates renal inflammation and fibrosis with reduces macrophage numbers, pro-inflammatory polarization and migration into unilateral ureteral obstruction (UUO) kidneys and unilateral ischemia reperfusion injury (IRI) models. Finally, we developed a selective boron containing HCK inhibitor which can reduce macrophage pro-inflammatory activity, proliferation, and migration in vitro, and attenuate kidney fibrosis in the UUO mice. The current study elucidates mechanisms downstream of HCK regulating macrophage activation and polarization via autophagy in CKD and identifies that selective HCK inhibitors could be potentially developed as a new therapy for renal fibrosis.


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The high-resolution video files of cell 3D-random migration are too large to upload as supplementary files and are available from the corresponding authors on reasonable request. All other relevant data supporting the key findings of this study are available within the article and its Supplementary Information files. Source data are provided with this paper.
Both male and female were included in the patient kidney biopsy collection. Please find more information on our previous paper PMID: 27452608 Lancet 2016; 388: 983-93.
All the race were included in the patient kidney biopsy collection. Please find more information on our previous paper PMID: No power analysis was performed to determine the sample size. For mice experiments, We used 8 mice (n=8) in each group. In vitro studies were performed with a minimum 3 (n=3) biological replication.
No data was excluded.
All experiments were repeated three times with similar results. Only the cytokine array only conducted once as the high cost.
For all the samples in mouse and cell experiments, they were allocated to different groups randomly.
All experiments in our study were blinded to investigators during group allocation. All quantification analyses for mouse and in vitro studies were also blinded for investigators.  All antibodies used in this study were commercial and validated by the manufacturer. Species and application validations and citations for primary antibodies can be found from the manufacturer's websites.
All cells were authenticated by short tandem repeat (STR) analysis.
All lines tested negative for mycoplasma using the Sigma Look-Out PCR Detection Kit.
No commonly misidentified cell lines were used in the study.
HCK exon3 loxp flanked 15 transgenic mouse at EuMMCR in Germany. C57BL/6J WT, CMV-Cre (#006054) and LysM-Cre (# 004781) mice were purchased from the Jackson Laboratory. All mice were maintained in our animal facility under controlled environmental conditions: 12/12 light/dark cycle, ambient temperature 20-25°C. More information can be found through their website: https:// icahn.mssm.edu/research/ccms. Mice at 8-10 week age were used for experiments in this study.